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Parasitology International Apr 2022Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important...
Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important defense strategies against parasites. Hence, parasites partly deal with oxidative stress by different mechanisms. Identifying resistance mechanisms of A. castellanii parasites against oxidative stress is important to achieve a new therapeutic approach. Thus, this study aimed to understand the resistance mechanisms of A. castellanii, against oxidative stress. Trophozoites of A. castellanii were treated with different concentrations of HO. The half maximal inhibitory concentration (IC) of HO was determined using the MTT assay. The induction of oxidative stress was confirmed by flow cytometer. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. The gene expression levels of CAT and SOD were measured by qRT-PCR. Furthermore, 3-amino-1:2:4-triazole (3-AT) and potassium cyanide (KCN) were used as specific inhibitors of CAT and SOD, respectively. Cell cycle assay and the apoptosis were evaluated by flow cytometer. The activities of SOD, CAT, GR, and GPx, showed an increase in oxidative stress. The cell cycle analysis revealed that most of the cellular population was in G0 and G1 phases. The apoptosis increased in oxidative stress conditions. Moreover, the apoptosis significantly increased after the specific inhibition of CAT and SOD under oxidative stress. The gene expression levels of CAT and SOD significantly increased under oxidative stress. A. castellanii can resist the host immune system through various mechanisms, including evoking its antioxidant enzymes. Therefore, by reducing or inhibiting the activity of the parasite's antioxidant enzymes such as SOD and CAT, it is possible to cope with A. castellanii.
Topics: Acanthamoeba castellanii; Animals; Antioxidants; Apoptosis; Catalase; Cell Cycle; Gene Expression Regulation, Enzymologic; Genotype; Glutathione Peroxidase; Glutathione Reductase; Hydrogen Peroxide; Inhibitory Concentration 50; Oxidative Stress; Superoxide Dismutase
PubMed: 34929407
DOI: 10.1016/j.parint.2021.102523 -
Parasitology Research Mar 2013During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst,...
During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.
Topics: Acanthamoeba castellanii; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Spores, Protozoan
PubMed: 23319266
DOI: 10.1007/s00436-012-3261-7 -
Acta Tropica Aug 2022Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other...
Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other parasitic protozoa, the available treatments show variable effectiveness in addition to high toxicity, which demands the development of new treatments. Positive results of 5-nitroindazole derivatives against several protozoa parasites suggest that these compounds may be a promising tool for the development of efficient antiparasitic drugs. In the present work we have evaluated the in vitro activity of ten 5-nitroindazole derivatives against Acanthamoeba castellanii trophozoites and cysts. To that end, AlamarBlue Assay Reagent® was used to determine the activity against trophozoites compared to the reference drug chlorhexidine digluconate. Cytotoxicity of the compounds was evaluated using Vero cells. The activity on cysts was evaluated by light microscopy and using a Neubauer chamber to quantifying cysts and presence of trophozoites, as an indication of cyst. Our results showed the effectiveness of the 5-nitroindazole derivatives tested against both trophozoites and cysts of A. castellani highlighting 5-nitroindazole derivative 8 which showed a 80% activity on cysts, which is higher than that of the reference drug. Moreover, 5-nitroindazole derivatives 8, 9 and 10 were more effective on trophozoites than the reference drug showing IC values lower than 5 µM. Taking together these results, these 5-nitroindazole derivatives specially compound 8, might be a promising alternative for the development of more efficient treatments against A. castellani infection.
Topics: Acanthamoeba castellanii; Animals; Chlorocebus aethiops; Humans; Indazoles; Trophozoites; Vero Cells
PubMed: 35618027
DOI: 10.1016/j.actatropica.2022.106538 -
Diagnostic Microbiology and Infectious... Oct 2023Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and...
Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.
Topics: Humans; Acanthamoeba castellanii; Rapid Diagnostic Tests; Acanthamoeba Keratitis; Cornea; Real-Time Polymerase Chain Reaction
PubMed: 37506594
DOI: 10.1016/j.diagmicrobio.2023.116014 -
Cellular and Molecular Life Sciences :... Apr 2021The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative...
The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii's response to oxidative stress. The role of the large TrxR, however, remains elusive.
Topics: Acanthamoeba castellanii; Antioxidants; Glutathione Disulfide; Glutathione Reductase; Humans; Oxidation-Reduction; Oxidative Stress; Thioredoxin-Disulfide Reductase; Thioredoxins
PubMed: 33599799
DOI: 10.1007/s00018-021-03786-x -
Parasite (Paris, France) 2021Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK)....
Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.
Topics: Acanthamoeba castellanii; Amebiasis; Biguanides; Cytochrome P-450 Enzyme System; Humans
PubMed: 34762043
DOI: 10.1051/parasite/2021074 -
Parasitology Research Jul 2020Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba...
Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.
Topics: Acanthamoeba castellanii; Amebiasis; Amebicides; Cell Line; Cell Survival; Humans; Inhibitory Concentration 50; Parasite Encystment; Quinazolinones; Structure-Activity Relationship
PubMed: 32476058
DOI: 10.1007/s00436-020-06710-7 -
The Journal of Eukaryotic Microbiology Mar 2018Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic...
Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, in vitro. Oleic acid markedly inhibited the growth of A. castellanii, with trophozoite viability of 57.4% at a concentration of 200 μM. Caspase-3 staining and annexin V assays showed that apoptotic death occurred in A. castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin-1β converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A. castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.
Topics: Acanthamoeba castellanii; Apoptosis; Autophagy; Caspase 3; Oleic Acid; Trophozoites
PubMed: 28787535
DOI: 10.1111/jeu.12454 -
Archives of Virology Feb 2022Here, we propose the creation of the family "Yaraviridae", a new taxon to classify a virus infecting Acanthamoeba castellanii cells. Recently, we described the discovery...
Here, we propose the creation of the family "Yaraviridae", a new taxon to classify a virus infecting Acanthamoeba castellanii cells. Recently, we described the discovery of a new virus infecting free-living amoebae, yaravirus, which has features that strongly differ from those of all other viruses of amoebae described to date. Yaravirus particles are about 80 nm in diameter and have a dsDNA genome of ~45 kbp containing 74 ORFs, most of which (>90%) have no homologs in current databases. Together, these data support the creation of a new species ("Yaravirus brasiliense"), a new viral genus (here proposed as "Yaravirus"), and a new viral family (here proposed as "Yaraviridae") to classify yaravirus and other related viruses that may be described in the future. All of them are to be included into the existing realm Varidnaviria and the kingdom Bamfordvirae, due to the presence of a major capsid protein containing a double jelly-roll fold.
Topics: Acanthamoeba castellanii; Capsid Proteins; DNA Viruses; Genome, Viral
PubMed: 35000005
DOI: 10.1007/s00705-021-05326-1 -
Translational Vision Science &... Sep 2023To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human...
PURPOSE
To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).
METHODS
Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO2]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.
RESULTS
A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO2 concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO2 and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO2.
CONCLUSIONS
Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.
TRANSLATIONAL RELEVANCE
This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.
Topics: Humans; Animals; Acanthamoeba castellanii; Nitric Oxide Donors; Hypochlorous Acid; NF-E2-Related Factor 2; TOR Serine-Threonine Kinases; Mammals
PubMed: 37768280
DOI: 10.1167/tvst.12.9.23